CN109996866A - Method for generating endothelial cell - Google Patents
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- CN109996866A CN109996866A CN201780074594.2A CN201780074594A CN109996866A CN 109996866 A CN109996866 A CN 109996866A CN 201780074594 A CN201780074594 A CN 201780074594A CN 109996866 A CN109996866 A CN 109996866A
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Abstract
本发明涉及一种用于产生内皮细胞的方法，其中按以下顺序进行步骤(a)在不形成胚状体的情况下从多能干细胞诱导含有内皮祖细胞的中胚层细胞群，和步骤(b)在存在RepSox的情况下培养含有内皮祖细胞的中胚层细胞群。根据本发明，从多能干细胞高效率地产生高质量的内皮细胞变成可能。通过本发明的方法产生的内皮细胞可用于例如产生心肌细胞层，并且预期用于治疗心脏疾病。通过将通过本发明的方法产生的内皮细胞与心肌细胞和壁细胞混合并然后培养该混合物，可产生心肌细胞层。The present invention relates to a method for producing endothelial cells, wherein step (a) inducing a population of mesodermal cells containing endothelial progenitor cells from pluripotent stem cells without forming embryoid bodies, and step (b) are performed in the following order ) Mesodermal cell populations containing endothelial progenitor cells were cultured in the presence of RepSox. According to the present invention, it becomes possible to efficiently generate high-quality endothelial cells from pluripotent stem cells. Endothelial cells produced by the methods of the present invention can be used, for example, to produce layers of cardiomyocytes, and are expected to be used in the treatment of cardiac disease. A cardiomyocyte layer can be produced by mixing endothelial cells produced by the method of the present invention with cardiomyocytes and parietal cells and then culturing the mixture.
Description
技术领域technical field
本发明涉及用于从多能干细胞产生高质量的内皮细胞的方法。The present invention relates to methods for generating high quality endothelial cells from pluripotent stem cells.
背景技术Background technique
已经开发了与再生疗法相关的技术创新，特别是用于产生多能干细胞的方法及用于诱导多能干细胞的分化的方法，使得已深入进行了针对生物组织的再生的研究。而且对于对单个生物的生命维持必不可少的心脏，认为再生医学技术可用于治疗心脏疾病。例如，已经尝试将从多能干细胞人工产生的心肌细胞应用于心脏疾病的治疗。此时心肌细胞不是单独使用，而是例如已经开发了用于形成含有心肌细胞的层状细胞构造的方法(专利公布1)，并且已经在具有心肌梗塞的模型动物中显示出由此获得的心肌层的有用性。Technological innovations related to regenerative therapy, in particular, methods for generating pluripotent stem cells and methods for inducing differentiation of pluripotent stem cells have been developed, so that research directed at regeneration of biological tissues has been intensively conducted. And for the heart, which is essential for the life-sustaining of a single organism, it is thought that regenerative medicine techniques can be used to treat heart disease. For example, attempts have been made to apply cardiomyocytes artificially generated from pluripotent stem cells to the treatment of heart disease. Cardiomyocytes are not used alone at this time, but, for example, a method for forming a lamellar cell structure containing cardiomyocytes has been developed (Patent Publication 1), and the myocardium thus obtained has been shown in model animals with myocardial infarction usefulness of layers.
在专利公布1中，通过将心肌细胞与内皮细胞、壁细胞和Flk /KDR阳性细胞组合并培养细胞来产生心肌层。尽管以上公布公开了作为细胞混合物制备多种细胞比如心肌细胞、内皮细胞和壁细胞的方法，但是从控制心肌层的质量或产生过程的观点来看，期望单独制备这些细胞。另外，当人工构建除血管以外的组织或器官时，预期内皮细胞被用作用于组织中形成的血管的材料。In Patent Publication 1, the myocardium is produced by combining cardiomyocytes with endothelial cells, parietal cells and Flk/KDR positive cells and culturing the cells. Although the above publication discloses a method of preparing various cells such as cardiomyocytes, endothelial cells and parietal cells as a cell mixture, it is desirable to prepare these cells individually from the viewpoint of controlling the quality of the myocardium or the production process. In addition, when tissues or organs other than blood vessels are artificially constructed, endothelial cells are expected to be used as a material for blood vessels formed in the tissue.
已知有几种用于产生内皮细胞的方法。一种用于从多能干细胞有效产生内皮细胞的方法为例如专利公布2中描述的方法。尽管该方法为在含有不同活性成分的多种培养基中逐步培养多能干细胞的方法，但仍然存在改善所获得的细胞质量的空间。Several methods are known for generating endothelial cells. A method for efficiently generating endothelial cells from pluripotent stem cells is, for example, the method described in Patent Publication 2. Although this method is a stepwise culture of pluripotent stem cells in various media containing different active components, there is still room for improving the quality of the cells obtained.
现有技术参考文献prior art references
专利公布Patent publication
专利公布1：WO 2012/133945Patent Publication 1: WO 2012/133945
专利公布2：WO 2014/192925Patent Publication 2: WO 2014/192925
发明概述SUMMARY OF THE INVENTION
本发明要解决的问题Problem to be solved by the present invention
为了使用内皮细胞作为用于产生如上所述的心肌层的材料并使用内皮细胞本身作为医学组合物或用于研究的材料，期望一种有效产生更高质量的内皮细胞的方法。本文使用的术语“高质量的内皮细胞”是指具有一种或多种以下特征的内皮细胞，比如获得的细胞数量高、纯度高(内皮细胞标志物的阳性率高)、分化阶段为同质的、具有极好的细胞增殖能力(例如幼年)、更不易受冷冻和解冻损伤的影响、和批次之间的差异小。In order to use endothelial cells as a material for producing the myocardium as described above and use endothelial cells themselves as a medical composition or a material for research, a method for efficiently producing endothelial cells of higher quality is desired. As used herein, the term "high-quality endothelial cells" refers to endothelial cells that have one or more of the following characteristics, such as a high number of cells obtained, high purity (high positivity for endothelial cell markers), and a homogeneous differentiation stage , has excellent cell proliferation ability (eg, juveniles), is less susceptible to freezing and thawing damage, and has small batch-to-batch variation.
本发明旨在解决常规产生方法所具有的问题，并且本发明的目的为提供一种用于产生高质量的内皮细胞的方法。The present invention aims to solve the problems of conventional production methods, and an object of the present invention is to provide a method for producing high-quality endothelial cells.
解决问题的方法way of solving the problem
作为解决上述问题的深入研究的结果，本发明人首次发现当从多能干细胞诱导内皮细胞时，通过在存在不含RepSox的培养基的情况下培养内皮祖细胞可获得大量的高纯度内皮细胞。进一步地，本发明人已经发现，通过以上方法获得的大多数内皮细胞为幼年内皮细胞，并且甚至在冷冻保存之后也保持高成活力和增殖率。本发明基于上述发现得以完成。As a result of intensive studies to solve the above-mentioned problems, the present inventors found for the first time that when endothelial cells are induced from pluripotent stem cells, a large amount of high-purity endothelial cells can be obtained by culturing endothelial progenitor cells in the presence of a medium without RepSox. Further, the present inventors have found that most endothelial cells obtained by the above method are juvenile endothelial cells and maintain high viability and proliferation rate even after cryopreservation. The present invention has been completed based on the above findings.
具体来说，本发明涉及：Specifically, the present invention relates to:
[1] 一种用于产生内皮细胞的方法，其包括按以下顺序进行：[1] A method for generating endothelial cells comprising performing in the following order:
(a) 在不形成胚状体的情况下从多能干细胞诱导含有内皮祖细胞的中胚层谱系细胞群；和(a) induction of mesoderm lineage cell populations containing endothelial progenitor cells from pluripotent stem cells without formation of embryoid bodies; and
(b) 在存在RepSox的情况下培养含有内皮祖细胞的中胚层谱系细胞群。(b) Mesodermal lineage cell populations containing endothelial progenitor cells were cultured in the presence of RepSox.
[2] 以上[1]的方法，其中多能干细胞为胚胎干细胞(ES细胞)或诱导性多能干细胞(iPS细胞)；[2] The method of [1] above, wherein the pluripotent stem cells are embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells);
[3] 以上[1]或[2]的方法，其中在步骤(a)中在以下培养基中依序培养多能干细胞：[3] The method of [1] or [2] above, wherein in step (a) pluripotent stem cells are sequentially cultured in the following medium:
(i) 含有激活素A的培养基，(i) a medium containing activin A,
(ii) 含有骨形态发生蛋白4的培养基，和(ii) medium containing bone morphogenetic protein 4, and
(iii) 含有血管内皮生长因子的培养基；(iii) a medium containing vascular endothelial growth factor;
[4] 以上[3]的方法，其中(ii)含有骨形态发生蛋白4的培养基进一步含有碱性成纤维细胞生长因子；[4] The method of [3] above, wherein (ii) the medium containing bone morphogenetic protein 4 further contains basic fibroblast growth factor;
[5] 以上[1]~[4]中任何一项的方法，其特征为在步骤(a)之后进一步包括(a') 从中胚层谱系细胞群分离内皮祖细胞；[5] The method of any one of the above [1] to [4], further comprising (a') isolating endothelial progenitor cells from the mesoderm lineage cell population after step (a);
[6] 以上[5]的方法，其中在步骤(a')中，激酶插入结构域受体阳性细胞被分离为内皮祖细胞；[6] The method of [5] above, wherein in step (a'), the kinase insertion domain receptor-positive cells are isolated as endothelial progenitor cells;
[7] 以上[1]~[6]中任何一项的方法，其中在步骤(b)之前，含有内皮祖细胞的中胚层谱系细胞群或分离的内皮祖细胞在没有RepSox的培养基中进行培养；[7] The method of any one of [1] to [6] above, wherein prior to step (b), the mesodermal lineage cell population containing endothelial progenitor cells or the isolated endothelial progenitor cells is performed in a medium without RepSox nourish;
[8] 以上[1]~[7]中任何一项的方法，其特征为在步骤(b)之后进一步包括(c) 冷冻内皮细胞；[8] The method of any one of the above [1] to [7], further comprising (c) freezing endothelial cells after step (b);
[9] 一种用于产生心肌层的方法，其包括：[9] A method for generating a myocardium comprising:
根据以上[1]~[8]中的任何一项定义的方法产生内皮细胞，和Endothelial cells are generated according to the method defined in any one of [1]~[8] above, and
将内皮细胞与心肌细胞和壁细胞混合以培养细胞；和mixing endothelial cells with cardiomyocytes and parietal cells to culture cells; and
[10] 含有激酶插入结构域受体阳性内皮祖细胞和RepSox的细胞组合物。[10] Cell composition containing kinase insertion domain receptor positive endothelial progenitor cells and RepSox.
发明效果Invention effect
根据本发明的方法，可从多能干细胞有效地产生高质量的内皮细胞。根据本发明的方法获得的内皮细胞可用于产生例如心肌层，并且预期用于治疗心脏疾病。进一步地，细胞可用作由内皮细胞的异常等引起的疾病的细胞模型，或者作为药物安全性评价的材料。另外，通过将通过本发明的方法获得的内皮细胞与心肌细胞和壁细胞混合并培养细胞，可产生心肌层。According to the method of the present invention, high-quality endothelial cells can be efficiently generated from pluripotent stem cells. Endothelial cells obtained according to the method of the present invention can be used to generate, for example, the myocardium, and are expected to be used in the treatment of cardiac diseases. Further, the cells can be used as cell models of diseases caused by abnormalities of endothelial cells or the like, or as materials for drug safety evaluation. In addition, by mixing endothelial cells obtained by the method of the present invention with cardiomyocytes and parietal cells and culturing the cells, a myocardium can be produced.
用于实施本发明的方式Modes for Carrying out the Invention
本发明将在下文详细说明。The present invention will be described in detail below.
本文使用的术语“多能干细胞”是指具有能够分化成多种细胞的多能性以及还具有自我增殖能力的干细胞，并且多能干细胞包括例如(但不限于)诱导性多能干细胞(iPS细胞)、胚胎干细胞(ES细胞)、种系干细胞(GS细胞)、胚胎生殖细胞(EG细胞)、源自通过核移植获得的克隆胚胎的胚胎干细胞(核移植ES细胞；ntES细胞)、融合干细胞等。优选的多能干细胞为iPS细胞或ES细胞，并且更优选的多能干细胞为iPS细胞。As used herein, the term "pluripotent stem cells" refers to stem cells that have pluripotency capable of differentiating into various cells and also have the ability to self-proliferate, and pluripotent stem cells include, for example, but not limited to, induced pluripotent stem cells (iPS cells). ), embryonic stem cells (ES cells), germline stem cells (GS cells), embryonic germ cells (EG cells), embryonic stem cells derived from cloned embryos obtained by nuclear transfer (nuclear transfer ES cells; ntES cells), fusion stem cells, etc. . Preferred pluripotent stem cells are iPS cells or ES cells, and more preferred pluripotent stem cells are iPS cells.
iPS细胞为一种体细胞来源的人工干细胞，其具有与ES细胞几乎相同的特性，例如分化多能性和通过自我复制的增殖能力，并且iPS细胞可通过将核酸或蛋白质形式的特定核重编程物质引入到体细胞中或通过用试剂增加核重编程物质的内源性mRNA和/或蛋白质的表达水平来产生(K. Takahashi和S. Yamanaka (2006), Cell, 126: 663-676; K.Takahashi等(2007), Cell, 131: 861-872; J. Yu等(2007), Science, 318: 1917-1920; 和M. Nakagawa等(2008), Nat. Biotechnol., 26: 101-106)。核重编程物质可为在ES细胞中特异性表达的基因或者在维持ES细胞的未分化状态中起重要作用的基因或其基因产物。该物质包括例如(但不特别限于) Oct3/4、Klf4、Klf1、Klf2、Klf5、Sox2、Sox1、Sox3、Sox15、Sox17、Sox18、c-Myc、L-Myc、N-Myc、TERT、SV40大T抗原、HPV16 E6、HPV16 E7、Bmil、Lin28、Lin28b、Nanog、Esrrb或Esrrg。当建立iPS细胞时，这些核重编程物质可组合使用。例如，该组合包括至少1种、2种或3种上述核重编程物质，和优选地该组合包括4种上述核重编程物质。iPS cells are artificial stem cells derived from somatic cells, which have almost the same properties as ES cells, such as differentiation pluripotency and proliferative ability through self-replication, and iPS cells can be reprogrammed by specific nuclei in the form of nucleic acids or proteins Substances are introduced into somatic cells or produced by increasing the expression level of endogenous mRNA and/or protein of nuclear reprogramming substances with agents (K. Takahashi and S. Yamanaka (2006), Cell , 126: 663-676; K. .Takahashi et al. (2007), Cell , 131: 861-872; J. Yu et al. (2007), Science , 318: 1917-1920; and M. Nakagawa et al. (2008), Nat. Biotechnol. , 26: 101-106 ). The nuclear reprogramming substance may be a gene specifically expressed in ES cells or a gene or gene product thereof that plays an important role in maintaining the undifferentiated state of ES cells. Such substances include, for example, but not limited to, Oct3/4, Klf4, Klf1, Klf2, Klf5, Sox2, Sox1, Sox3, Sox15, Sox17, Sox18, c-Myc, L-Myc, N-Myc, TERT, SV40 T antigen, HPV16 E6, HPV16 E7, Bmil, Lin28, Lin28b, Nanog, Esrrb or Esrrg. When establishing iPS cells, these nuclear reprogramming substances can be used in combination. For example, the combination includes at least 1, 2 or 3 of the above-described nuclear reprogramming substances, and preferably the combination includes 4 of the above-described nuclear reprogramming substances.
作为细胞系建立的人iPS细胞系可用于本发明的实施方案中。优选的人iPS细胞系为(但不特别限于) ChiPSC7、ChiPSC11、ChiPSC12、ChiPSC19、ChiPSC20、ChiPSC21、ChiPSC22、ChiPSC23、201B7、201B7-Ff、253G1、253G4、1201C1、1205D1、1210B2和836B3，和更优选的人iPS细胞系为ChiPSC12。上述人iPS细胞系可从Cellartis或iPS Academia Japan,Inc.或Center for iPS Cell Research and Application, Kyoto University获得。Human iPS cell lines established as cell lines can be used in embodiments of the present invention. Preferred human iPS cell lines are (but not particularly limited to) ChiPSC7, ChiPSC11, ChiPSC12, ChiPSC19, ChiPSC20, ChiPSC21, ChiPSC22, ChiPSC23, 201B7, 201B7-Ff, 253G1, 253G4, 1201C1, 1205D1, 1210B2 and 836B3, and more preferably The human iPS cell line is ChiPSC12. The above-mentioned human iPS cell lines can be obtained from Cellartis or iPS Academia Japan, Inc. or the Center for iPS Cell Research and Application, Kyoto University.
ES细胞为从哺乳动物比如人或小鼠的早期胚胎(例如胚泡)的内细胞团建立，并且具有分化多能性和通过自我复制的增殖能力的干细胞。1981年在小鼠中发现了ES细胞(M.J. Evans和M. H. Kaufman (1981), Nature292: 154-156)，并且随后还在灵长类动物比如人和猴中建立了ES细胞。ES cells are stem cells established from the inner cell mass of early embryos (eg, blastocysts) of mammals such as humans or mice, and possessing pluripotency to differentiate and the ability to proliferate by self-replication. ES cells were discovered in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292: 154-156) and subsequently established in primates such as humans and monkeys.
ES细胞可通过从受试动物的受精卵的胚泡中提取内细胞团并在成纤维细胞的饲养细胞上培养内细胞团来建立。另外，通过传代培养维持细胞可通过使用补充有物质比如LIF或bFGF的培养基来进行。用于建立和维持人和猴的ES细胞的方法描述于例如H.Suemori等(2006), Biochem. Biophys. Res. Commun., 345: 926-932; H. Kawasaki等(2002), Proc. Natl. Acad. Sci. USA, 99: 1580-1585等中。另外，一些研究机构分配ES细胞。例如，作为人ES细胞系的KhES-1、KhES-2和KhES-3可从Institute for FrontierMedical Sciences, Kyoto University (Kyoto, Japan)获得。ES cells can be established by extracting the inner cell mass from blastocysts of fertilized eggs of a subject animal and culturing the inner cell mass on feeder cells of fibroblasts. In addition, maintenance of cells by subculture can be performed by using a medium supplemented with substances such as LIF or bFGF. Methods for establishing and maintaining human and monkey ES cells are described, for example, in H. Suemori et al. (2006), Biochem. Biophys. Res. Commun. , 345: 926-932; H. Kawasaki et al. (2002), Proc. Natl . Acad. Sci. USA , 99: 1580-1585 et al. Additionally, some research institutions distribute ES cells. For example, KhES-1, KhES-2, and KhES-3, which are human ES cell lines, are available from the Institute for Frontier Medical Sciences, Kyoto University (Kyoto, Japan).
多能干细胞的培养通过优选地使根据任何方法制备的多能干细胞经受在合适的培养容器/培养基底中的粘附培养来进行。这里，粘附培养是指在细胞粘附于培养容器/培养基底的状态下培养细胞。在粘附培养中，未形成胚状体(EB)。通过使用涂有细胞可粘附的物质的培养容器/培养基底进行粘附培养。细胞可粘附的物质包括例如各种细胞外基质(胶原蛋白、明胶、层连蛋白、纤连蛋白、玻连蛋白、巢蛋白、硫酸乙酰肝素蛋白聚糖等)、其改变的产物或修饰的产物、聚赖氨酸及其组合。优选地，本发明中使用Matrigel (注册商标)或Synthemax (注册商标)，其每种均为含有多种细胞外基质的组合物。Cultivation of pluripotent stem cells is carried out by preferably subjecting pluripotent stem cells prepared according to any method to adherent culture in a suitable culture vessel/culture substrate. Here, adherent culture refers to culturing cells in a state where cells adhere to the culture vessel/culture substrate. In adherent cultures, embryoid bodies (EBs) were not formed. Adherent culture is performed by using culture vessels/culture substrates coated with a cell-adherent substance. Substances to which cells can adhere include, for example, various extracellular matrices (collagen, gelatin, laminin, fibronectin, vitronectin, nestin, heparan sulfate proteoglycans, etc.), altered products or modified forms thereof. Products, polylysine, and combinations thereof. Preferably, Matrigel (registered trademark) or Synthemax (registered trademark), each of which is a composition containing a plurality of extracellular matrices, is used in the present invention.
对于本发明的该步骤和其它培养步骤中使用的培养容器/培养基底，可使用具有任何种类的材料或形状的那些，只要其不抑制细胞的维持、成活力、分化、成熟或自我复制。培养容器/培养基底的形状也没有限制，并且可使用具有任何形状的那些，比如瓶、板、皿、袋和温育器。可使用各种市售可得的培养容器/培养基底。进一步地，可使用提供有中空丝的培养容器、或培养基底比如珠粒进行培养。For the culture vessel/culture substrate used in this and other culture steps of the present invention, those of any kind of material or shape can be used as long as it does not inhibit cell maintenance, viability, differentiation, maturation or self-replication. The shape of the culture vessel/culture substrate is also not limited, and those having any shape can be used, such as bottles, plates, dishes, bags and incubators. Various commercially available culture vessels/culture substrates can be used. Further, culturing can be performed using a culture vessel provided with hollow fibers, or a culture substrate such as beads.
作为基础培养基，可使用可用于培养动物细胞的培养基。例如，RPMI 1640培养基、DEF-CS培养基、培养基199、MCDB131培养基、IMDM、EMEM、αMEM、DMEM、Ham F12培养基、Fischer培养基、其混合培养基等用作基础培养基。进一步地，也使用无Xeno培养基或合成培养基(化学限定培养基)。可使用作为用于培养多能干细胞的培养基销售的市售可得的培养基，例如Stem Fit (注册商标)、mTeSR1、Essentia18 (商标)等。优选地，DEF-CS培养基用作基础培养基。培养基可添加有血清，或者可不含血清。任选地，可向其中添加例如白蛋白、转铁蛋白、生长因子、KSR、N2补充剂、B27补充剂、脂肪酸、胰岛素、胶原蛋白前体、脂类、氨基酸、维生素、2-巯基乙醇、3'-硫代甘油、微量元素、抗生素、抗氧化剂、缓冲剂、无机盐等。As the basal medium, a medium that can be used for culturing animal cells can be used. For example, RPMI 1640 medium, DEF-CS medium, medium 199, MCDB131 medium, IMDM, EMEM, αMEM, DMEM, Ham F12 medium, Fischer medium, mixed medium thereof and the like are used as the basal medium. Further, Xeno-free medium or synthetic medium (chemically defined medium) was also used. Commercially available media sold as media for culturing pluripotent stem cells, such as Stem Fit (registered trademark), mTeSR1, Essentia18 (trademark), and the like can be used. Preferably, DEF-CS medium is used as basal medium. The medium may be serum-supplemented, or may be serum-free. Optionally, for example, albumin, transferrin, growth factors, KSR, N2 supplements, B27 supplements, fatty acids, insulin, collagen precursors, lipids, amino acids, vitamins, 2-mercaptoethanol, 3'-thioglycerol, trace elements, antibiotics, antioxidants, buffers, inorganic salts, etc.
将细胞以例如0.5-20 x 104个细胞/cm2，优选地1-10 x 104个细胞/cm2的密度接种至涂有细胞可粘附的物质的培养容器中，并将细胞在合适的培养基中进行培养。培养期包括例如12小时或更长时间，优选地24小时或更长时间，和更优选地3天或更长时间，但是预期根据细胞确定合适的培养期。另外，在培养期间可适当地进行培养基更换或传代培养。Cells are seeded at a density of, for example, 0.5-20 x 10 4 cells/cm 2 , preferably 1-10 x 10 4 cells/cm 2 into culture vessels coated with a cell-adherent substance, and the cells are placed in cultured in a suitable medium. The culturing period includes, for example, 12 hours or more, preferably 24 hours or more, and more preferably 3 days or more, but it is expected that a suitable culturing period will be determined according to the cells. In addition, medium exchange or subculture may be appropriately performed during the culture.
用于从培养容器中分离本发明的已经受粘附培养的多能干细胞和其它细胞的方法包括物理方法、使用螯合剂的方法、使用具有蛋白酶活性和/或胶原酶活性的分离溶液(例如TrypLE (商标) Select、Accutase (注册商标)、Accumax (注册商标)等)的酶促方法，及其组合。优选地，在通过酶促方法解离多能干细胞的集落之后，进行使细胞物理经受精细分散的方法。通常，将已紧靠所用的培养容器培养至80%汇合的多能干细胞经受分离操作。Methods for isolating adherent cultured pluripotent stem cells and other cells of the present invention from culture vessels include physical methods, methods using chelating agents, using isolation solutions having protease activity and/or collagenase activity (eg, TrypLE). (trademark) Enzymatic methods of Select, Accutase (registered trademark), Accumax (registered trademark), etc.), and combinations thereof. Preferably, after dissociating the colonies of pluripotent stem cells by enzymatic methods, the method of physically subjecting the cells to fine dispersion is carried out. Typically, pluripotent stem cells that have been cultured to 80% confluence in close proximity to the culture vessel used are subjected to the isolation procedure.
(1) 本发明的用于产生内皮细胞的方法(1) The method for producing endothelial cells of the present invention
(a) 在不形成胚状体的情况下从多能干细胞诱导含有内皮祖细胞的中胚层谱系细胞群的步骤(a) Steps to induce a population of mesodermal lineage cells containing endothelial progenitor cells from pluripotent stem cells without formation of embryoid bodies
该步骤为从多能干细胞诱导分化成含有内皮祖细胞的中胚层谱系细胞群的步骤。在本发明的一个优选实施方案中从iPS细胞或ES细胞，和在特别优选的实施方案中从iPS细胞诱导含有内皮祖细胞的中胚层谱系细胞群。在该步骤中，通过优选地进行接触培养，可在不形成胚状体的情况下诱导以上细胞群。This step is a step of inducing differentiation from pluripotent stem cells into a population of mesodermal lineage cells containing endothelial progenitor cells. In a preferred embodiment of the invention a population of mesodermal lineage cells containing endothelial progenitor cells is induced from iPS cells or ES cells, and in a particularly preferred embodiment from iPS cells. In this step, by preferably performing contact culture, the above cell population can be induced without forming embryoid bodies.
本文使用的术语“中胚层谱系细胞群”意指含有中胚层细胞本身和/或通过从中胚层细胞分化产生的细胞的细胞群。通过从中胚层细胞分化产生的细胞包括成血管细胞、间充质干细胞、造血干细胞、内皮祖细胞、心脏祖细胞等。The term "mesodermal lineage cell population" as used herein means a cell population containing mesodermal cells themselves and/or cells generated by differentiation of mesodermal cells. Cells generated by differentiation of mesoderm cells include hemangioblasts, mesenchymal stem cells, hematopoietic stem cells, endothelial progenitor cells, cardiac progenitor cells, and the like.
本文使用的术语“内皮祖细胞”意指其分化指向内皮细胞的细胞。内皮祖细胞可通过分析转录因子或细胞表面抗原的表达模式来确认。例如，单独或组合测量转录因子或细胞表面抗原的表达模式，其中其表达水平未被检测到或低(即使在诱导分化之前被检测到)，并且在诱导分化之后显著增加。有效用于确认内皮祖细胞的标志物包括例如激酶插入结构域受体(KDR)、FOXF1、BMP4、MOX1、SDF1等。优选地，内皮细胞祖细胞为表达KDR的细胞。KDR为一种分子，其也称为血管内皮生长因子受体-2：VEGFR-2或Flk-1作为另一个名称，并且在血管形成的过程中起重要作用。The term "endothelial progenitor cells" as used herein means cells whose differentiation is directed towards endothelial cells. Endothelial progenitor cells can be identified by analyzing expression patterns of transcription factors or cell surface antigens. For example, the expression patterns of transcription factors or cell surface antigens are measured, alone or in combination, where their expression levels are undetectable or low (even if detected prior to induction of differentiation) and significantly increase after induction of differentiation. Markers useful for identifying endothelial progenitor cells include, for example, kinase insertion domain receptor (KDR), FOXF1, BMP4, MOX1, SDF1, and the like. Preferably, the endothelial cell progenitor cells are KDR-expressing cells. KDR is a molecule that is also known as Vascular Endothelial Growth Factor Receptor-2: VEGFR-2 or Flk-1 as another name, and plays an important role in the process of blood vessel formation.
该步骤(在不形成胚状体的情况下从多能干细胞诱导含有内皮祖细胞的中胚层谱系细胞群的步骤)没有特别限制，并且可从已知方法中选择合适的方法。用于本发明的优选方法包括这样的方法，即，其包括在存在激活素A和/或骨形态发生蛋白4 (BMP4)的情况下培养多能干细胞以提供中胚层细胞。更优选地，举例说明通过依序进行以下3步培养从多能干细胞诱导中胚层细胞的方法：“(i) 在含有激活素A的培养基中培养”，“(ii) 在含有BMP4的培养基中培养”和“(iii) 在含有血管内皮生长因子(VEGF)的培养基中培养。这种3步培养将在下文详细说明。This step (a step of inducing a population of mesodermal lineage cells containing endothelial progenitor cells from pluripotent stem cells without forming embryoid bodies) is not particularly limited, and an appropriate method can be selected from known methods. Preferred methods for use in the present invention include methods comprising culturing pluripotent stem cells in the presence of activin A and/or bone morphogenetic protein 4 (BMP4) to provide mesodermal cells. More preferably, a method for inducing mesoderm cells from pluripotent stem cells by culturing the following 3 steps in sequence: "(i) culturing in a medium containing activin A", "(ii) culturing in a BMP4-containing medium" is exemplified. Culture in medium" and "(iii) in medium containing vascular endothelial growth factor (VEGF). This 3-step culture will be described in detail below.
(i) 在含有激活素A的培养基中培养(i) Culture in medium containing activin A
作为本发明的一个实施方案，在含有激活素A的培养基中培养细胞之前，通过用细胞外基质以夹层法培养多能干细胞可有效诱导中胚层细胞。Matrigel可用作细胞外基质。具体来说，将多能干细胞以例如1-20 x 104个细胞/cm2，优选地2-15 x 104个细胞/cm2的密度接种至涂有Matrigel的培养容器中，并使细胞经受在例如DEF-CS培养基中粘附培养2-3天。之后，将培养基更换为添加有稀释至例如1:10-1:300、优选地1:20-1:150和更优选地1:40-1:80的Matrigel的培养基，并将细胞进一步培养16-24小时，其中全部多能干细胞被Matrigel包被。由此获得的Matrigel包被的多能干细胞的培养基更换为含有激活素A的培养基。As one embodiment of the present invention, mesodermal cells can be efficiently induced by culturing pluripotent stem cells in a sandwich method with an extracellular matrix before culturing the cells in a medium containing activin A. Matrigel can be used as extracellular matrix. Specifically, pluripotent stem cells are seeded into Matrigel-coated culture vessels at a density of, for example, 1-20 x 10 4 cells/cm 2 , preferably 2-15 x 10 4 cells/cm 2 , and the cells are allowed to Subject to adherent culture in eg DEF-CS medium for 2-3 days. Afterwards, the medium is changed to medium supplemented with Matrigel diluted eg to 1:10-1:300, preferably 1:20-1:150 and more preferably 1:40-1:80, and the cells are further Cultured for 16-24 hours, all of the pluripotent stem cells were coated with Matrigel. The medium of the thus obtained Matrigel-coated pluripotent stem cells was replaced with a medium containing activin A.
添加至培养基中的激活素A的浓度为例如10-1000 ng/mL，优选地为25-500 ng/mL，和更优选地为50-200 ng/mL。另外，可在该范围内向培养基中添加其它生长因子等，使得不损害本发明的效果，并且优选地在不存在BMP4和VEGF的情况下进行这种添加。The concentration of Activin A added to the culture medium is, for example, 10-1000 ng/mL, preferably 25-500 ng/mL, and more preferably 50-200 ng/mL. In addition, other growth factors and the like may be added to the medium within this range so as not to impair the effect of the present invention, and such addition is preferably performed in the absence of BMP4 and VEGF.
作为基础培养基，可使用可用于培养动物细胞的培养基。例如，RPMI 1640培养基、DEF-CS培养基、培养基199、MCDB131培养基、IMDM、EMEM、αMEM、DMEM、Ham F12培养基、Fischer培养基、其混合培养基等用作基础培养基。可使用作为用于培养多能干细胞的培养基销售的市售可得的培养基。优选地，RPMI 1640培养基用作基础培养基。培养基可添加有血清，或者可不含血清。任选地，可向其中添加例如白蛋白、转铁蛋白、生长因子、KSR、N2补充剂、B27补充剂、脂肪酸、胰岛素、胶原蛋白前体、脂类、氨基酸、维生素、2-巯基乙醇、3'-硫代甘油、微量元素、抗生素、抗氧化剂、缓冲剂、无机盐等。As the basal medium, a medium that can be used for culturing animal cells can be used. For example, RPMI 1640 medium, DEF-CS medium, medium 199, MCDB131 medium, IMDM, EMEM, αMEM, DMEM, Ham F12 medium, Fischer medium, mixed medium thereof and the like are used as the basal medium. Commercially available media sold as media for culturing pluripotent stem cells can be used. Preferably, RPMI 1640 medium is used as basal medium. The medium may be serum-supplemented, or may be serum-free. Optionally, for example, albumin, transferrin, growth factors, KSR, N2 supplements, B27 supplements, fatty acids, insulin, collagen precursors, lipids, amino acids, vitamins, 2-mercaptoethanol, 3'-thioglycerol, trace elements, antibiotics, antioxidants, buffers, inorganic salts, etc.
优选的培养基包括添加有L-谷氨酰胺、B27补充剂和激活素A的RPMI 1640培养基。此处，代替添加至以上培养基或本发明的另一种培养基中的L-谷氨酰胺，可添加L-丙氨酰L-谷氨酰胺二肽或GlutaMAX (商标)。GlutaMAX (商标)含有L-丙氨酰L-谷氨酰胺二肽。由于具有稳定结构的L-丙氨酰L-谷氨酰胺二肽在储存或培养期间不会以与L-谷氨酰胺相同的方式分解以形成有毒的氨，因此L-丙氨酰L-谷氨酰胺二肽适合于细胞培养。Preferred media include RPMI 1640 medium supplemented with L-glutamine, B27 supplement and activin A. Here, instead of L-glutamine added to the above medium or another medium of the present invention, L-alanyl L-glutamine dipeptide or GlutaMAX (trademark) may be added. GlutaMAX (trademark) contains L-alanyl L-glutamine dipeptide. Since L-alanyl L-glutamine dipeptide, which has a stable structure, does not break down in the same way as L-glutamine to form toxic ammonia during storage or culture, L-alanyl L-glutamine Aminoamide dipeptides are suitable for cell culture.
使多能干细胞经受在上述培养基中的粘附培养。培养温度为例如(但不限于) 30℃-40℃，和优选地为37℃。培养在含有CO2的空气的气氛下进行，和CO2浓度优选地为2-8%。培养时间为例如6小时-5天，和优选地为12-48小时。Pluripotent stem cells were subjected to adherent culture in the above-mentioned medium. The culture temperature is, for example, but not limited to, 30°C to 40°C, and preferably 37°C. The cultivation is performed in an atmosphere of air containing CO 2 , and the CO 2 concentration is preferably 2-8%. The culturing time is, for example, 6 hours to 5 days, and preferably 12 to 48 hours.
(ii) 在含有BMP4的培养基中培养(ii) Culture in medium containing BMP4
已经在含有激活素A的培养基中培养的多能干细胞随后经受在含有BMP4的培养基中培养。该培养可以与以上(i)的培养相同的方式进行，或者可以与以上培养不同的方式进行。优选地，用细胞外基质进行粘附培养，例如使用Matrigel进行粘附培养。在通过与以上培养相同的方法进行培养的情况下，可更换培养基，并然后可在同一容器中继续培养。Pluripotent stem cells that had been cultured in Activin A-containing medium were then subjected to culture in BMP4-containing medium. The culturing may be performed in the same manner as the culturing of (i) above, or may be performed in a different manner than the culturing above. Preferably, the adherent culture is carried out with an extracellular matrix, for example using Matrigel. In the case of culturing by the same method as the above culturing, the medium can be replaced, and then the culturing can be continued in the same vessel.
添加至培养基中的BMP4的浓度为例如0.5-200 ng/mL，优选地为1-100 ng/mL，和更优选地为2-50 ng/mL。The concentration of BMP4 added to the medium is, for example, 0.5-200 ng/mL, preferably 1-100 ng/mL, and more preferably 2-50 ng/mL.
在本发明的一个优选实施方案中，将碱性成纤维细胞生长因子(bFGF)进一步添加至培养基中。添加至可用于本实施方案的培养基中的bFGF的浓度为例如0.5-200 ng/mL，优选地为1-100 ng/mL，和更优选地为2-50 ng/mL。进一步地，可在该范围内向培养基中添加其它生长因子等，使得不损害由本发明呈现的效果，并且优选地在不存在激活素A和VEGF的情况下进行这种添加。In a preferred embodiment of the present invention, basic fibroblast growth factor (bFGF) is further added to the culture medium. The concentration of bFGF added to the medium useful in this embodiment is, for example, 0.5-200 ng/mL, preferably 1-100 ng/mL, and more preferably 2-50 ng/mL. Further, other growth factors and the like may be added to the medium within this range so as not to impair the effects exhibited by the present invention, and such addition is preferably performed in the absence of activin A and VEGF.
用于该培养的培养基可通过适当地组合与上述那些相同的基础培养基和各种组分来制备。优选的培养基包括添加有L-谷氨酰胺、B27补充剂、bFGF和BMP4的RPMI 1640培养基。The medium used for this culture can be prepared by appropriately combining the same basal medium and various components as those described above. Preferred media include RPMI 1640 medium supplemented with L-glutamine, B27 supplement, bFGF and BMP4.
培养温度为例如(但不限于) 30℃-40℃，和优选地为37℃。培养在含有CO2的空气的气氛下进行，和CO2浓度优选地为2-8%。培养时间为例如12小时-5天，和优选地为2-4天。The culture temperature is, for example, but not limited to, 30°C to 40°C, and preferably 37°C. The cultivation is performed in an atmosphere of air containing CO 2 , and the CO 2 concentration is preferably 2-8%. The culturing time is, for example, 12 hours to 5 days, and preferably 2 to 4 days.
(iii) 在含有VEGF的培养基中培养(iii) Cultivation in VEGF-containing medium
在以上(ii)的培养之后，使细胞经受在含有VEGF的培养基中培养。该培养也可以与以上(i)和(ii)的培养相同的方式进行。优选地，进行用细胞外基质的粘附培养。Following the culturing of (ii) above, the cells were subjected to culturing in a medium containing VEGF. The culturing can also be performed in the same manner as the culturing of (i) and (ii) above. Preferably, adherent culture with extracellular matrix is performed.
可用于该培养的培养基中的VEGF的浓度为例如10-2000 ng/mL，和优选地为50-500 ng/mL。进一步地，可在该范围内向培养基中添加其它生长因子等，使得不损害由本发明呈现的效果，并且优选地在不存在激活素A和BMP4的情况下进行这种培养。The concentration of VEGF in the medium that can be used for this culture is, for example, 10-2000 ng/mL, and preferably 50-500 ng/mL. Further, other growth factors and the like may be added to the medium within this range so as not to impair the effects exhibited by the present invention, and such culture is preferably performed in the absence of activin A and BMP4.
用于该培养的培养基也可通过适当地组合与上述那些相同的基础培养基和各种组分来制备。优选的培养基包括添加有L-谷氨酰胺、B27补充剂和VEGF的RPMI 1640培养基。The medium used for this culture can also be prepared by appropriately combining the same basal medium and various components as those described above. Preferred media include RPMI 1640 medium supplemented with L-glutamine, B27 supplement and VEGF.
培养温度为例如(但不限于) 30℃-40℃，和优选地为37℃。培养在含有CO2的空气的气氛下进行，和CO2浓度优选地为2-8%。培养时间为例如6小时-5天，和优选地为12-48小时。The culture temperature is, for example, but not limited to, 30°C to 40°C, and preferably 37°C. The cultivation is performed in an atmosphere of air containing CO 2 , and the CO 2 concentration is preferably 2-8%. The culturing time is, for example, 6 hours to 5 days, and preferably 12 to 48 hours.
通过该培养，获得含有内皮祖细胞的中胚层谱系细胞群。此处，内皮祖细胞的比率(KDR阳性率)为例如总细胞数的40%或更多，优选地50%或更多，和更优选地60%或更多。Through this culture, a population of mesodermal lineage cells containing endothelial progenitor cells is obtained. Here, the ratio of endothelial progenitor cells (KDR positive ratio) is, for example, 40% or more, preferably 50% or more, and more preferably 60% or more of the total cell number.
(a’) 从中胚层谱系细胞群中分离内皮祖细胞的步骤(a’) Steps to isolate endothelial progenitor cells from mesoderm lineage cell populations
该步骤为从上述步骤中获得的含有内皮祖细胞的中胚层谱系细胞群中分离内皮祖细胞的步骤。此处，当上述步骤中获得的含有内皮祖细胞的中胚层谱系细胞群中的内皮祖细胞的比率(KDR阳性率)已足够高时，也可不进行该步骤。优选的是该步骤(a’)在步骤(a)之后进行。This step is a step of isolating endothelial progenitor cells from the mesodermal lineage cell population containing endothelial progenitor cells obtained in the above step. Here, when the ratio of endothelial progenitor cells (KDR positive rate) in the mesodermal lineage cell population containing endothelial progenitor cells obtained in the above step is sufficiently high, this step may not be performed. It is preferred that this step (a') is carried out after step (a).
在该步骤中分离内皮祖细胞通过用荧光激活细胞分选术(FACS)、磁激活细胞分选术(MACS)等选择性地捕获表达上述内皮祖细胞标志物的细胞，并然后收集捕获的细胞来进行。在上述分离方法中，有利的是使用与内皮祖细胞标志物特异性结合的抗体。因此，例如，与KDR或在内皮祖细胞的表面上表达的其它标志物结合的抗体或其片段用于本发明。In this step, endothelial progenitor cells are isolated by selectively capturing cells expressing the aforementioned endothelial progenitor cell markers by fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), etc., and then collecting the captured cells to proceed. In the above isolation methods, it is advantageous to use antibodies that specifically bind to markers of endothelial progenitor cells. Thus, for example, antibodies or fragments thereof that bind to KDR or other markers expressed on the surface of endothelial progenitor cells are useful in the present invention.
具体来说，使含有内皮祖细胞的细胞群与适当标记的抗体接触，所述抗体与内皮祖细胞标志物特异性结合，例如抗KDR抗体。之后，通过收集标记的细胞实现内皮祖细胞的分离。例如，可通过使与荧光标记的抗体接触的细胞群经受经FACS的分离来分离抗体结合的细胞。另外，在使用以磁性材料标记的抗体的情况下，可使用MACS等。进一步地，以上抗体可与另一种抗体(二抗)组合，所述二抗与附着于以上抗体的标记物或抗体本身特异性结合。在这种情况下，通过使用附着于二抗的标记物进行细胞的分离。市售可得的抗体可用作以上抗KDR抗体或二抗。例如，可使用“与PE荧光标记物结合的抗KDR抗体”和“包埋抗PE抗体的磁珠”，但不特别限于此。Specifically, the endothelial progenitor cell-containing cell population is contacted with an appropriately labeled antibody that specifically binds to an endothelial progenitor cell marker, eg, an anti-KDR antibody. Afterwards, isolation of endothelial progenitor cells is achieved by collecting labeled cells. For example, antibody-bound cells can be isolated by subjecting a population of cells in contact with the fluorescently labeled antibody to separation by FACS. In addition, when an antibody labeled with a magnetic material is used, MACS or the like can be used. Further, the above antibody may be combined with another antibody (secondary antibody) that specifically binds to the label attached to the above antibody or the antibody itself. In this case, isolation of cells is performed by using a label attached to the secondary antibody. Commercially available antibodies can be used as the above anti-KDR antibodies or secondary antibodies. For example, "anti-KDR antibody conjugated with PE fluorescent label" and "magnetic beads embedded with anti-PE antibody" can be used, but are not particularly limited thereto.
分离的内皮祖细胞的比率(KDR阳性率)为例如70%或更多，优选地为80%或更多，和更优选地为90%或更多。The ratio of isolated endothelial progenitor cells (KDR positive rate) is, for example, 70% or more, preferably 80% or more, and more preferably 90% or more.
分离的内皮祖细胞可直接分化成内皮细胞，或者内皮祖细胞可在维持培养一次之后分化成内皮细胞，同时保持内皮祖细胞的分化状态。The isolated endothelial progenitor cells can be directly differentiated into endothelial cells, or the endothelial progenitor cells can be differentiated into endothelial cells after one maintenance culture, while maintaining the differentiated state of the endothelial progenitor cells.
在内皮祖细胞经受维持培养同时保持内皮祖细胞的分化状态的情况下，可以与上述步骤中的培养相同的方式进行维持培养。具体来说，通过使用细胞外基质比如Matrigel的粘附培养进行维持培养。用于该维持培养的培养基也可通过适当地组合与上述那些相同的基础培养基与各种组分来制备。可使用作为用于培养内皮细胞的培养基(例如用于增殖内皮细胞的培养基等)销售的市售可得的培养基。优选的培养基包括添加有L-谷氨酰胺、B27补充剂和VEGF的RPMI 1640培养基。培养基可进一步含有抗生素(比如青霉素或链霉素)或ROCK抑制剂。ROCK抑制剂没有特别限制，只要抑制剂可抑制Rho激酶(ROCK)的功能，并且抑制剂包括例如Y-27632。Y-27632的浓度为例如1-500 μM，优选地为3-200 μM，和更优选地为10-50 μM。进一步地，可在该范围内向培养基中添加其它生长因子等，使得不损害由本发明呈现的效果，并且优选地在不存在以下详述的RepSox的情况下进行这种维持培养。In the case where the endothelial progenitor cells are subjected to maintenance culture while maintaining the differentiated state of the endothelial progenitor cells, the maintenance culture can be performed in the same manner as the culture in the above-mentioned steps. Specifically, maintenance culture is performed by adherence culture using an extracellular matrix such as Matrigel. The medium used for this maintenance culture can also be prepared by appropriately combining the same basal medium as those described above with various components. A commercially available medium sold as a medium for culturing endothelial cells (eg, a medium for proliferating endothelial cells, etc.) can be used. Preferred media include RPMI 1640 medium supplemented with L-glutamine, B27 supplement and VEGF. The medium may further contain antibiotics (such as penicillin or streptomycin) or ROCK inhibitors. The ROCK inhibitor is not particularly limited as long as the inhibitor can inhibit the function of Rho kinase (ROCK), and the inhibitor includes, for example, Y-27632. The concentration of Y-27632 is, for example, 1-500 μM, preferably 3-200 μM, and more preferably 10-50 μM. Further, other growth factors and the like may be added to the medium within this range so as not to impair the effects exhibited by the present invention, and such maintenance culture is preferably performed in the absence of RepSox detailed below.
维持培养的培养时间包括例如24小时或更长时间，优选地2天或更长时间，和更优选地3天或更长时间，并且预期根据细胞的状态或培养条件确定合适的培养时间。另外，在培养期间可适当地进行培养基更换或传代培养。The culture time for maintaining the culture includes, for example, 24 hours or more, preferably 2 days or more, and more preferably 3 days or more, and it is expected that an appropriate culture time is determined according to the state of the cells or the culture conditions. In addition, medium exchange or subculture may be appropriately performed during the culture.
通常，在该维持培养步骤中，内皮祖细胞的数量没有显示出显著的增加或减少。另外，维持培养几乎不降低内皮祖细胞的比率(KDR阳性率)。例如，在维持培养进行3天之后，内皮祖细胞的比率(KDR阳性率)为70%或更多，优选地为80%或更多，和更优选地为90%或更多。Typically, the number of endothelial progenitor cells does not show a significant increase or decrease during this maintenance culture step. In addition, the maintenance culture hardly decreased the ratio of endothelial progenitor cells (KDR positive ratio). For example, after the maintenance culture is performed for 3 days, the ratio of endothelial progenitor cells (KDR positive rate) is 70% or more, preferably 80% or more, and more preferably 90% or more.
(b) 在存在RepSox的情况下培养含有内皮祖细胞的中胚层谱系细胞群的步骤(b) Procedure for culturing a population of mesodermal lineage cells containing endothelial progenitor cells in the presence of RepSox
该步骤为在存在RepSox的情况下培养在上述步骤中获得的含有内皮祖细胞的中胚层谱系细胞群的步骤。在进行步骤(a')的情况下，在该步骤中，在存在RepSox的情况下培养内皮祖细胞。This step is a step of culturing the mesodermal lineage cell population containing endothelial progenitor cells obtained in the above step in the presence of RepSox. Where step (a') is performed, in this step, endothelial progenitor cells are cultured in the presence of RepSox.
随后使上述步骤中获得的含有内皮祖细胞的中胚层谱系细胞群或内皮祖细胞经受在含有RepSox的培养基中的培养。该培养步骤可以与上述步骤中的培养相同的方式进行，或者可以与上述步骤中的培养不同的方式进行。优选地，进行用细胞外基质的粘附培养。在以与上述步骤中的培养相同的方式进行培养的情况下，更换培养基，并然后可在同一容器中继续培养。在使用涂有Matrigel的培养容器的情况下，例如含有内皮祖细胞的中胚层谱系细胞群或内皮祖细胞以0.5-10 x 104个细胞/cm2，和优选地1-5 x 104个细胞/cm2的密度接种，并且细胞在含有RepSox的培养基中培养。由于在该步骤中通过使用RepSox首次使内皮祖细胞分化成内皮细胞，优选的是在该步骤(b)之前将含有内皮祖细胞的中胚层谱系细胞群或分离的内皮祖细胞在不含RepSox的培养基中进行培养。The endothelial progenitor cell-containing mesoderm lineage cell population or endothelial progenitor cells obtained in the above steps are then subjected to culture in a medium containing RepSox. This culturing step may be performed in the same manner as the culturing in the above-mentioned steps, or may be performed in a different manner than the culturing in the above-mentioned steps. Preferably, adherent culture with extracellular matrix is performed. In the case of culturing in the same manner as the culturing in the above-mentioned steps, the medium is replaced, and then the culturing can be continued in the same vessel. In the case of using Matrigel-coated culture vessels, such as a population of mesodermal lineage cells containing endothelial progenitor cells or endothelial progenitor cells at 0.5-10 x 10 4 cells/cm 2 , and preferably 1-5 x 10 4 cells Cells/ cm2 were seeded at a density and cells were cultured in medium containing RepSox. Since endothelial progenitor cells are differentiated into endothelial cells for the first time by using RepSox in this step, it is preferred to separate the population of mesodermal lineage cells containing endothelial progenitor cells or isolated endothelial progenitor cells in RepSox-free prior to this step (b). cultured in medium.
RepSox (CAS号446859-33-2)为低分子量化合物，其也称为E-616452、SJN2511、Alk5抑制剂II、TGF-βRI激酶抑制剂II等作为另一个名称。RepSox为ALK5 (TGF-β受体之一)的强选择性抑制剂，其通过抑制ALK5来抑制TGF-β的功能。RepSox为TGF-β抑制剂之一。此处，转化生长因子(TGF)-β为具有许多特征的天然存在的生长因子之一，其在组织发育、细胞分化、胚胎生长等中起重要作用。RepSox (CAS No. 446859-33-2) is a low molecular weight compound, which is also known as E-616452, SJN2511, Alk5 Inhibitor II, TGF-βRI Kinase Inhibitor II, etc. as another name. RepSox is a potent and selective inhibitor of ALK5 (one of the TGF-β receptors), which inhibits the function of TGF-β by inhibiting ALK5. RepSox is one of TGF-β inhibitors. Here, transforming growth factor (TGF)-β is one of naturally occurring growth factors having many characteristics, which play an important role in tissue development, cell differentiation, embryo growth, and the like.
SB431542 (CAS号301836-41-9)为低分子量化合物，其抑制ALK4、ALK5和ALK7 (其各自均为TGF-β受体)。SB431542通过抑制上述受体组来抑制TGF-β的功能。SB431542为TGF-β抑制剂之一。SB431542 (CAS No. 301836-41-9) is a low molecular weight compound that inhibits ALK4, ALK5 and ALK7 (each of which is a TGF-beta receptor). SB431542 inhibits the function of TGF-β by inhibiting the above-mentioned receptor group. SB431542 is one of TGF-β inhibitors.
可用于该步骤的RepSox的浓度没有特别限制，只要RepSox可实现期望的效果，比如高效诱导内皮细胞。添加至用于该步骤的培养基中的RepSox的浓度为例如0.5-100 μM，优选地为1-50 μM，和更优选地为3-30 μM。The concentration of RepSox that can be used in this step is not particularly limited as long as RepSox can achieve the desired effect, such as the efficient induction of endothelial cells. The concentration of RepSox added to the medium used for this step is, for example, 0.5-100 μM, preferably 1-50 μM, and more preferably 3-30 μM.
用于该步骤的培养基也可通过适当地组合与上述那些相同的基础培养基与各种组分来制备。优选的培养基包括添加有RepSox的用于增殖内皮细胞的培养基。The medium used for this step can also be prepared by appropriately combining the same basal medium as those described above with various components. Preferred media include media for proliferating endothelial cells supplemented with RepSox.
在存在RepSox的情况下的培养时间包括例如24小时-14天，和优选地为2-7天，并且预期根据RepSox的浓度确定合适的培养时间。另外，RepSox的浓度可适当地变化。The incubation time in the presence of RepSox includes, for example, 24 hours to 14 days, and preferably 2 to 7 days, and it is expected that the appropriate incubation time will be determined according to the concentration of RepSox. In addition, the concentration of RepSox can be appropriately varied.
在存在RepSox的情况下培养含有内皮祖细胞的中胚层谱系细胞群或内皮祖细胞，从而可促进内皮祖细胞的增殖，并且结果是可大量获得表达内皮细胞标志物的内皮细胞。另外，在该步骤中获得的内皮细胞具有一种或多种以下特征，比如获得的细胞数量高、纯度高(内皮细胞标志物的阳性率高)、分化阶段为同质的、具有极好的细胞增殖能力(例如幼年)、更不易受冷冻和解冻损伤的影响、和批次之间的差异小。Culturing a population of mesodermal lineage cells or endothelial progenitor cells containing endothelial progenitor cells in the presence of RepSox can promote the proliferation of endothelial progenitor cells, and as a result, endothelial cells expressing endothelial cell markers can be obtained in large quantities. In addition, the endothelial cells obtained in this step have one or more of the following characteristics, such as high number of cells obtained, high purity (high positive rate of endothelial cell markers), homogeneous differentiation stage, excellent Cell proliferative capacity (eg, juvenile), less susceptible to freezing and thawing damage, and small batch-to-batch variation.
本文使用的术语“内皮细胞”意指表达至少一种内皮细胞标志物比如PE-CAM(CD31)、VE-钙粘蛋白(CD144)、内皮糖蛋白(CD105)和冯维勒布兰德因子(vWF)的细胞。此处，表达CD31的细胞为优选，和表达CD31和CD144两者的细胞为更优选。通过本发明获得的内皮细胞含有CD31阳性细胞，其比率为例如70%或更多，优选地为80%或更多，更优选地为90%或更多，但本发明不特别限于此。The term "endothelial cell" as used herein means expressing at least one endothelial cell marker such as PE-CAM (CD31), VE-cadherin (CD144), endoglin (CD105) and von Willebrand factor ( vWF) cells. Here, cells expressing CD31 are preferred, and cells expressing both CD31 and CD144 are more preferred. The endothelial cells obtained by the present invention contain CD31-positive cells at a ratio of, for example, 70% or more, preferably 80% or more, more preferably 90% or more, but the present invention is not particularly limited thereto.
内皮细胞可根据其分化阶段更精细地分类。在内皮细胞中，更加未分化的细胞称为“幼年内皮细胞”，和具有分化进展的细胞称为“成熟内皮细胞”。内皮细胞的分化阶段可通过分析转录因子或细胞表面抗原的表达模式来确认。具体来说，单独或组合测量转录因子或细胞表面抗原的表达模式，其中其表达水平根据内皮细胞的分化进展而显著变化。有效确认内皮细胞的分化阶段的标志物包括例如上述内皮细胞标志物(CD31、CD144、CD105、vWF)和CD34。CD34为造血干细胞或内皮祖细胞的标志物。另外，CD34也在幼年内皮细胞上表达。表达CD31和CD34两者的细胞(下文中CD31+/CD34+细胞)为(但不特别限于)幼年内皮细胞的一个实例。通过本发明获得的内皮细胞含有大量的幼年内皮细胞，并且例如细胞含有70%或更多、优选地80%或更多和更优选地90%或更多的比率的CD31+/CD34+细胞，但本发明不特别限于此。Endothelial cells can be classified more finely according to their stage of differentiation. Among endothelial cells, the more undifferentiated cells are called "juvenile endothelial cells", and the cells that have progressed in differentiation are called "mature endothelial cells". The differentiation stage of endothelial cells can be confirmed by analyzing the expression patterns of transcription factors or cell surface antigens. Specifically, expression patterns of transcription factors or cell surface antigens, whose expression levels vary significantly according to the progression of endothelial cell differentiation, are measured alone or in combination. Markers effective for confirming the differentiation stage of endothelial cells include, for example, the aforementioned endothelial cell markers (CD31, CD144, CD105, vWF) and CD34. CD34 is a marker of hematopoietic stem cells or endothelial progenitor cells. In addition, CD34 is also expressed on juvenile endothelial cells. Cells expressing both CD31 and CD34 (hereinafter CD31+/CD34+ cells) are, but not particularly limited to, one example of juvenile endothelial cells. The endothelial cells obtained by the present invention contain a large number of juvenile endothelial cells, and for example the cells contain CD31+/CD34+ cells at a ratio of 70% or more, preferably 80% or more and more preferably 90% or more, but the present The invention is not particularly limited to this.
根据本发明的用于产生内皮细胞的方法，可产生各种内皮细胞比如血管内皮细胞、淋巴管内皮细胞或角膜内皮细胞。According to the method for producing endothelial cells of the present invention, various endothelial cells such as vascular endothelial cells, lymphatic endothelial cells or corneal endothelial cells can be produced.
通过继续培养内皮细胞可获得更大量的内皮细胞。作为用于该步骤的培养基，可使用通过适当地组合基础培养基和各种组分制备的培养基，并且优选的是该步骤在不存在RepSox的情况下进行。可使用作为用于培养内皮细胞的培养基(例如用于增殖内皮细胞的培养基)销售的市售可得的培养基。例如，内皮细胞可通过使用用于增殖内皮细胞的培养基，并且继续培养同时每1-3天进行培养基更换和传代培养来产生。由此产生的内皮细胞可长期保持内皮细胞的特性。Larger amounts of endothelial cells can be obtained by continuing to culture the endothelial cells. As a medium for this step, a medium prepared by appropriately combining a basal medium and various components can be used, and it is preferable that this step is performed in the absence of RepSox. Commercially available media sold as media for culturing endothelial cells (eg, media for proliferating endothelial cells) can be used. For example, endothelial cells can be generated by using a medium for proliferating endothelial cells, and continuing the culture with medium change and subculture every 1-3 days. The resulting endothelial cells maintain the properties of endothelial cells for a long time.
另外，在该步骤(b)中未观察到内皮细胞标志物的大量减少。内皮细胞可保持(但不特别限于) CD31阳性率超过60%，和优选地CD31阳性率超过80%，甚至在开始诱导分化之后30天时。In addition, no substantial reduction of endothelial cell markers was observed in this step (b). Endothelial cells may maintain, but are not particularly limited to, CD31 positivity of more than 60%, and preferably CD31 positivity of more than 80%, even 30 days after initiation of induction of differentiation.
(c) 冷冻内皮细胞的步骤(c) Steps for freezing endothelial cells
该步骤为在步骤(b)之后进行的步骤，并且为冷冻在上述一系列步骤中获得的内皮细胞的步骤。通过将内皮细胞悬浮于含有冷冻保护剂的溶液中，将该悬浮液分配于合适的储存容器中，并根据适当的方法冷冻储存容器来进行内皮细胞的冷冻。This step is a step performed after the step (b), and is a step of freezing the endothelial cells obtained in the above-mentioned series of steps. Freezing of endothelial cells is carried out by suspending endothelial cells in a solution containing a cryoprotectant, dispensing the suspension into a suitable storage container, and freezing the storage container according to an appropriate method.
作为可用于冷冻内皮细胞的步骤的冷冻保护剂，可使用任何冷冻保护剂，只要冷冻保护剂不抑制细胞的维持、成活力、分化、成熟或自我复制。例如可使用甘油、乙二醇、二甲基亚砜、蔗糖、葡萄糖、聚乙烯吡咯烷酮、海藻糖等。还可使用各种市售可得的冷冻保护剂。例如，由Nippon Zenyaku Kogyo Co., Ltd制造的STEM-CELLBANKER (注册商标)，和优选地由Nippon Zenyaku Kogyo Co., Ltd.制造的CELLBANKER (注册商标)用作冷冻保护剂。例如，内皮细胞悬浮于(但不特别限于) CELLBANKER中，使得其浓度为0.5-10 x 106/mL，和优选地为1-5 x 106/mL，并将悬浮液分配于储存容器中。As a cryoprotectant that can be used in the step of freezing endothelial cells, any cryoprotectant can be used as long as the cryoprotectant does not inhibit the maintenance, viability, differentiation, maturation or self-replication of the cells. For example, glycerol, ethylene glycol, dimethyl sulfoxide, sucrose, glucose, polyvinylpyrrolidone, trehalose and the like can be used. Various commercially available cryoprotectants can also be used. For example, STEM-CELLBANKER (registered trademark) manufactured by Nippon Zenyaku Kogyo Co., Ltd., and preferably CELLBANKER (registered trademark) manufactured by Nippon Zenyaku Kogyo Co., Ltd. are used as cryoprotectants. For example, endothelial cells are suspended in (but not particularly limited to) CELLBANKER such that their concentration is 0.5-10 x 10 6 /mL, and preferably 1-5 x 10 6 /mL, and the suspension is dispensed into storage containers .
作为储存容器，可使用具有任何材料和形状的那些储存容器，只要该容器不抑制细胞的维持、成活力、分化、成熟或自我复制。储存容器的形状也没有特别限制，并且可使用具有任何形状的容器，比如小瓶、瓶或袋。可使用各种市售可得的储存容器。例如小瓶用作储存容器。As storage containers, those of any material and shape can be used as long as the container does not inhibit the maintenance, viability, differentiation, maturation or self-replication of cells. The shape of the storage container is also not particularly limited, and a container having any shape, such as a vial, a bottle, or a bag, can be used. Various commercially available storage containers can be used. For example vials are used as storage containers.
作为冷冻方法，进行任何方法，只要该方法不抑制细胞的维持、成活力、分化、成熟或自我复制。例如进行缓慢冷冻，但不特别限于此。也可用程序化的冷冻机进行缓慢冷冻，并且也可使用冷冻处理的容器比如BICELL进行缓慢冷冻。As the freezing method, any method is performed as long as the method does not inhibit the maintenance, viability, differentiation, maturation or self-replication of cells. For example, slow freezing is performed, but is not particularly limited thereto. Slow freezing can also be performed with a programmed freezer, and slow freezing can also be performed using a freezer processing container such as a BICELL.
冷冻的内皮细胞可储存于液氮、保持在-80℃的深度冷冻机等中。当细胞储存于液氮中时，内皮细胞可储存例如24小时或更长时间，优选地3天或更长时间，和更优选地2周或更长时间。Frozen endothelial cells can be stored in liquid nitrogen, deep freezers maintained at -80°C, or the like. When the cells are stored in liquid nitrogen, endothelial cells can be stored, for example, for 24 hours or longer, preferably 3 days or longer, and more preferably 2 weeks or longer.
作为用于解冻冷冻的内皮细胞的方法，进行任何方法，只要该方法不抑制细胞的维持、成活力、分化、成熟或自我复制。例如将细胞用温热至37℃的水浴温热并解冻，但不特别限于此。解冻的内皮细胞的成活力为60%或更高，优选地为70%或更高，和更优选地为80%或更高。As a method for thawing frozen endothelial cells, any method is performed as long as the method does not inhibit the maintenance, viability, differentiation, maturation or self-replication of the cells. For example, the cells are warmed and thawed in a water bath warmed to 37°C, but not particularly limited thereto. The viability of the thawed endothelial cells is 60% or higher, preferably 70% or higher, and more preferably 80% or higher.
解冻的内皮细胞的培养可以与本发明中的其它细胞的培养相同的方式进行。具体来说，培养优选地通过用细胞外基质的粘附培养来进行。用于该培养的培养基也可通过适当地组合与上述那些相同的基础培养基和各种组分来制备。优选的培养基包括用于增殖内皮细胞的培养基。培养时间包括例如24小时或更长时间，优选地3天或更长时间，和更优选地7天或更长时间，并且预期根据需要的细胞量确定合适的培养时间。另外，可在培养期间适当地进行培养基更换或传代培养。例如，在培养进行7天的情况下，内皮祖细胞增加至例如1.1倍或更多，优选地1.3倍或更多，和更优选地1.5倍或更多。另外，内皮细胞的比率(CD31阳性率)几乎不降低。例如，在培养进行7天之后，内皮细胞的比率(CD31阳性率)为80%或更多，优选地为90%或更多，和更优选地为95%或更多。The culture of thawed endothelial cells can be performed in the same manner as the culture of other cells in the present invention. Specifically, the culture is preferably carried out by adherence culture with an extracellular matrix. The medium used for this culture can also be prepared by appropriately combining the same basal medium and various components as those described above. Preferred media include media used to proliferate endothelial cells. The culturing time includes, for example, 24 hours or more, preferably 3 days or more, and more preferably 7 days or more, and it is expected that an appropriate culturing time will be determined according to the desired cell amount. In addition, medium exchange or subculture can be appropriately performed during the culture. For example, in the case where the culture is performed for 7 days, the endothelial progenitor cells are increased by, for example, 1.1 times or more, preferably 1.3 times or more, and more preferably 1.5 times or more. In addition, the endothelial cell ratio (CD31 positive ratio) hardly decreased. For example, after culturing for 7 days, the ratio of endothelial cells (CD31 positive rate) is 80% or more, preferably 90% or more, and more preferably 95% or more.
(2) 本发明的用于产生心肌层的方法(2) The method for producing a myocardium of the present invention
通过上述本发明获得的内皮细胞可用于产生含有内皮细胞作为组分的心肌层。例如，日本专利特许公报第2012-210156号描述了一种用于产生心肌层的方法，其中使Flk/KDR阳性细胞与心肌细胞、内皮细胞和壁细胞混合以形成层。心肌层可按照日本专利特许公报第2012-210156号中描述的方法，使用本发明中产生的内皮细胞来产生，但本发明不特别限于此。The endothelial cells obtained by the present invention described above can be used to produce a myocardium containing endothelial cells as a component. For example, Japanese Patent Laid-Open No. 2012-210156 describes a method for producing a myocardium in which Flk/KDR-positive cells are mixed with cardiomyocytes, endothelial cells, and parietal cells to form a layer. The myocardium can be produced using the endothelial cells produced in the present invention according to the method described in Japanese Patent Laid-Open Publication No. 2012-210156, but the present invention is not particularly limited thereto.
本文使用的术语“心肌层”是指由形成心脏或血管的各种细胞组成的层状细胞构造，其中细胞经细胞间结合彼此连接。此处，形成心脏或血管的各种细胞包括心肌细胞、内皮细胞和壁细胞。下文将说明按照日本专利特许公报第2012-210156号中描述的方法产生心肌层的方法。The term "myocardium" as used herein refers to a layered cellular structure composed of various cells that form the heart or blood vessels, wherein the cells are connected to each other by intercellular bonds. Here, various cells forming the heart or blood vessels include cardiomyocytes, endothelial cells, and parietal cells. Hereinafter, a method of producing the myocardium according to the method described in Japanese Patent Laid-Open No. 2012-210156 will be explained.
<产生心肌细胞的步骤><Steps for generating cardiomyocytes>
该步骤为从多能干细胞产生心肌细胞的步骤，其通过已知方法实现。心肌细胞可通过例如按照WO 2012/133954中描述的方法培养多能干细胞来获得，但不特别限于此。This step is a step of generating cardiomyocytes from pluripotent stem cells, which is achieved by a known method. Cardiomyocytes can be obtained by, for example, culturing pluripotent stem cells according to the method described in WO 2012/133954, but not particularly limited thereto.
<产生FLK阳性细胞和壁细胞的步骤><Procedure for producing FLK-positive cells and parietal cells>
FLK阳性细胞可通过按照任何方法，例如Yamashita等(2000), Nature, 408: 92-96中描述的方法培养多能干细胞来获得。另外，通过基于相同参考文献的描述培养FLK阳性细胞，可获得内皮细胞和壁细胞的细胞混合物。进一步地，可通过使用与壁细胞标志物特异性结合的抗体来分离壁细胞。此处，壁细胞意指呈现出与血管壁细胞(来自其外部的血管内皮细胞周围的细胞)或其祖细胞等同的特性的细胞。FLK-positive cells can be obtained by culturing pluripotent stem cells according to any method, eg, as described in Yamashita et al. (2000), Nature , 408: 92-96. In addition, by culturing FLK-positive cells based on the description of the same reference, a cell mixture of endothelial cells and parietal cells can be obtained. Further, parietal cells can be isolated by using antibodies that specifically bind to markers of parietal cells. Here, the parietal cells mean cells exhibiting properties equivalent to vascular wall cells (cells surrounding vascular endothelial cells from the outside thereof) or progenitor cells thereof.
<产生心肌层的步骤><Procedure for generating myocardium>
FLK阳性细胞在温敏性培养容器，例如由CellSeed Inc.制造的UpCell等上培养。在细胞培养1-7天之后，例如3天之后，将通过本发明的方法制备的内皮细胞和壁细胞的细胞混合物和心肌细胞以适当的量加入到培养容器中。将细胞混合物在含有血管内皮生长因子(VEGF)的培养基中培养1-10天，例如4天，从而形成层状细胞构造。可通过使培养容器在室温下静置，从培养容器中取出由此形成的心肌层，从而可收集心肌层。进一步地，根据需要，层压产生的心肌层。FLK-positive cells are cultured on a temperature-sensitive culture vessel such as UpCell manufactured by CellSeed Inc. or the like. After the cells are cultured for 1-7 days, for example, after 3 days, the cell mixture of endothelial cells and parietal cells and cardiomyocytes prepared by the method of the present invention are added to the culture vessel in appropriate amounts. The cell mixture is cultured in a medium containing vascular endothelial growth factor (VEGF) for 1-10 days, eg, 4 days, to form a lamellar cell structure. The myocardium thus formed can be collected from the culture vessel by allowing the culture vessel to stand at room temperature, and then removing the myocardium thus formed. Further, the resulting myocardium is laminated as needed.
由此产生的心肌层可用于治疗心脏疾病，例如心力衰竭、心肌梗塞、缺血性心脏病、心肌炎、各种心肌病或其它应用。例如，对于心脏疾病比如心肌梗塞，放置心肌层以覆盖期望的心脏部位，从而进行治疗。心肌层被带至心脏组织，以促进心脏功能的恢复。The resulting myocardium can be used to treat cardiac diseases such as heart failure, myocardial infarction, ischemic heart disease, myocarditis, various cardiomyopathy, or other applications. For example, for cardiac diseases such as myocardial infarction, the myocardium is placed to cover the desired heart site for treatment. The myocardium is brought to the heart tissue to facilitate recovery of heart function.
根据本发明，除了用于产生内皮细胞的方法和用于产生心肌层的方法之外，还提供通过该方法产生的内皮细胞和通过该方法产生的心肌层。According to the present invention, in addition to the method for producing endothelial cells and the method for producing myocardium, endothelial cells produced by this method and myocardium produced by this method are also provided.
(3) 本发明的细胞组合物(3) Cell composition of the present invention
本发明的细胞组合物含有激酶插入结构域受体(KDR)阳性内皮祖细胞和RepSox，并且该组合物用于体外内皮细胞的产生。The cellular composition of the present invention contains kinase insertion domain receptor (KDR) positive endothelial progenitor cells and RepSox, and the composition is used for the generation of endothelial cells in vitro.
本发明的细胞组合物中含有的KDR阳性内皮祖细胞可通过按照已知方法分化多能干细胞来制备。例如，可通过使用多能干细胞比如ES细胞或iPS细胞作为材料，并且在本发明的用于产生内皮细胞的以上方法中进行步骤(a)，或者进行步骤(a)和步骤(b)，来获得内皮祖细胞。可根据需要进一步进行以上步骤(a')。此处，细胞组合物中含有的KDR阳性内皮祖细胞的含量为例如(但不特别限于)为KDR阳性的细胞的70%或更多，优选地80%或更多，和更优选地90%或更多。The KDR-positive endothelial progenitor cells contained in the cell composition of the present invention can be prepared by differentiating pluripotent stem cells according to known methods. For example, by using pluripotent stem cells such as ES cells or iPS cells as a material, and performing step (a), or performing both steps (a) and (b) in the above method for generating endothelial cells of the present invention, Obtain endothelial progenitor cells. The above step (a') may be further carried out as required. Here, the content of KDR-positive endothelial progenitor cells contained in the cell composition is, for example, but not particularly limited to, 70% or more, preferably 80% or more, and more preferably 90% of the KDR-positive cells Or more.
以上细胞组合物中含有的RepSox的浓度没有特别限制，只要可实现期望的效果比如高效诱导内皮细胞。RepSox的浓度为例如0.5-100 μM，优选地为1-50 μM，和更优选地为3-30 μM。The concentration of RepSox contained in the above cell composition is not particularly limited as long as the desired effect such as high-efficiency induction of endothelial cells can be achieved. The concentration of RepSox is, for example, 0.5-100 μM, preferably 1-50 μM, and more preferably 3-30 μM.
上述细胞组合物可含有通过适当地组合与本发明的其它步骤中使用的那些相同的基础培养基和各种组分制备的培养基。优选的培养基包括添加有RepSox的用于增殖内皮细胞的培养基。The cell composition described above may contain a medium prepared by appropriately combining the same basal medium and various components as those used in the other steps of the present invention. Preferred media include media for proliferating endothelial cells supplemented with RepSox.
将上述细胞组合物培养例如24小时-14天，和优选地2-7天，从而诱导内皮祖细胞分化成内皮细胞，从而可获得表达内皮细胞标志物的内皮细胞。预期根据使用的RepSox的浓度确定合适的培养时间。The above cell composition is cultured, for example, for 24 hours to 14 days, and preferably for 2 to 7 days, to induce the differentiation of endothelial progenitor cells into endothelial cells, thereby obtaining endothelial cells expressing endothelial cell markers. It is expected that the appropriate incubation time will be determined according to the concentration of RepSox used.
实施例Example
通过以下实施例更具体地描述本发明，但不旨在使本发明的范围限于这些实施例。The present invention is described in more detail by the following examples, which are not intended to limit the scope of the present invention to these examples.
实施例1：从iPS细胞分化成血管内皮细胞Example 1: Differentiation from iPS cells to vascular endothelial cells
按照培养基附带的说明书，在DEF-CS培养基(Cellartis)中培养人iPS细胞(ChiPSC12品系) (Cellartis)。Human iPS cells (ChiPSC12 strain) (Cellartis) were cultured in DEF-CS medium (Cellartis) according to the instructions accompanying the medium.
为了有效地诱导中胚层细胞的分化，按照以下程序用Matrigel包被iPS细胞(Matrigel夹层法)。首先，将TrypLE (商标) Select (Life technologies)添加至已传代培养的iPS细胞中，并将细胞在37℃下温育3-7分钟。通过吸液将分离的细胞收集为单细胞，并计数细胞数量。接下来，将细胞以6 x 104个细胞/cm2的密度接种至涂有Matrigel (商标)(Corning)的培养容器中，并在DEF-CS培养基中培养2-3天。之后，将培养基更换为添加有稀释至1:60的Matrigel的DEF-CS培养基，并将细胞再培养16-24小时，从而用Matrigel包被细胞的上层。To efficiently induce differentiation of mesodermal cells, iPS cells were coated with Matrigel (Matrigel sandwich method) according to the following procedure. First, TrypLE (trademark) Select (Life technologies) was added to subcultured iPS cells, and the cells were incubated at 37°C for 3-7 minutes. The detached cells were collected as single cells by pipetting and the number of cells was counted. Next, cells were seeded at a density of 6 x 104 cells/ cm2 into Matrigel (trademark) (Corning)-coated culture vessels and cultured in DEF-CS medium for 2-3 days. After that, the medium was changed to DEF-CS medium supplemented with Matrigel diluted to 1:60, and the cells were cultured for an additional 16-24 hours to coat the upper layer of cells with Matrigel.
按照以下程序使获得的Matrigel包被的iPS细胞分化成中胚层细胞。首先，将Matrigel包被的iPS细胞的培养基更换为添加有100 ng/mL激活素A (R&D)、2 mM GlutaMAX(商标) (Thermo Fisher Scientific)和B27补充剂(不含胰岛素) (Gibco)的RPMI 1640培养基(Gibco) (该时间点定义为开始诱导分化=第0天)，并将细胞培养24小时(第1天)。接下来，将培养基更换为添加有10 ng/mL人骨形态发生蛋白4 (hBMP4) (R&D)、10 ng/mL人碱性成纤维细胞生长因子(hbFGF) (Peprotech)、2 mM GlutaMAX和B27补充剂的RPMI 1640培养基，并将细胞培养3天(第4天)。之后，将培养基更换为添加有100 ng/mL血管内皮生长因子(VEGF) (Peprotech)、2 mM GlutaMAX和B27补充剂的RPMI 1640培养基，并将细胞培养过夜(第5天)。此处，作为用光学显微镜观察所培养的细胞的结果，从第0天至第5天不能确认胚状体的形成。The obtained Matrigel-coated iPS cells were differentiated into mesoderm cells according to the following procedure. First, the medium of Matrigel-coated iPS cells was changed to supplemented with 100 ng/mL Activin A (R&D), 2 mM GlutaMAX (trademark) (Thermo Fisher Scientific), and B27 supplement (without insulin) (Gibco) of RPMI 1640 medium (Gibco) (this time point was defined as initiation of induction of differentiation = day 0), and cells were cultured for 24 hours (day 1). Next, the medium was changed to one supplemented with 10 ng/mL human bone morphogenetic protein 4 (hBMP4) (R&D), 10 ng/mL human basic fibroblast growth factor (hbFGF) (Peprotech), 2 mM GlutaMAX, and B27 Supplemented RPMI 1640 medium and cells were cultured for 3 days (day 4). After that, the medium was changed to RPMI 1640 medium supplemented with 100 ng/mL vascular endothelial growth factor (VEGF) (Peprotech), 2 mM GlutaMAX and B27 supplement, and the cells were cultured overnight (day 5). Here, as a result of observing the cultured cells with an optical microscope, the formation of embryoid bodies could not be confirmed from the 0th day to the 5th day.
在第5天用磁激活细胞分选术[MACS(注册商标), Miltenyi biotech]从培养的细胞中分离激酶插入结构域受体(KDR)阳性细胞，从而分离血管内皮祖细胞。为了分离KDR阳性细胞，将结合有PE荧光标记物的抗KDR抗体(Miltenyi biotech)用作用于标记细胞的一抗，并用包埋抗PE抗体的磁珠(Miltenyi biotech)收集用一抗标记的细胞。当用流式细胞术测量通过MACS分离的细胞中存在的KDR阳性细胞的比率时，KDR阳性率为89-99%。将获得的细胞以2.7 x 104个细胞/cm2的密度接种至涂有Matrigel的培养容器中，并在添加有B27补充剂、50单位/mL青霉素、50 μg/mL链霉素、2 mM GlutaMAX、20 μM Y-27632和100 ng/mLVEGF的RPMI 1640培养基中培养16-24小时(第6天)。进一步地，将第6天的培养基更换为添加有B27补充剂、50单位/mL青霉素、50 μg/mL链霉素、2 mM GlutaMAX和100 ng/mL VEGF的RPMI 1640培养基，并将细胞培养2天(第8天)。Vascular endothelial progenitor cells were isolated by isolating kinase insertion domain receptor (KDR)-positive cells from the cultured cells using magnetic activated cell sorting [MACS (registered trademark), Miltenyi biotech] on day 5. To isolate KDR-positive cells, an anti-KDR antibody conjugated with a PE fluorescent marker (Miltenyi biotech) was used as the primary antibody for labeling cells, and the cells labeled with the primary antibody were collected with anti-PE antibody-embedded magnetic beads (Miltenyi biotech). . When the ratio of KDR-positive cells present in cells isolated by MACS was measured by flow cytometry, the KDR-positive rate was 89-99%. The obtained cells were seeded at a density of 2.7 x 104 cells/ cm2 into Matrigel-coated culture vessels and were supplemented with B27 supplement, 50 units/mL penicillin, 50 μg/mL streptomycin, 2 mM GlutaMAX, 20 μM Y-27632 and 100 ng/mL VEGF in RPMI 1640 medium for 16-24 hours (day 6). Further, the medium on day 6 was changed to RPMI 1640 medium supplemented with B27 supplement, 50 units/mL penicillin, 50 μg/mL streptomycin, 2 mM GlutaMAX, and 100 ng/mL VEGF, and cells were incubated Culture for 2 days (day 8).
在第8天收集细胞，并悬浮于以表1中列出的浓度添加有RepSox的用于增殖内皮细胞的培养基(Promocell)中。之后，将细胞以2 x 104个细胞/cm2的密度接种至培养容器中，并培养3天(第11天)。此处，另一种TGF-β抑制剂SB431542用作阴性对照。Cells were harvested on day 8 and suspended in Medium for Proliferating Endothelial Cells (Promocell) supplemented with RepSox at the concentrations listed in Table 1 . After that, cells were seeded into culture vessels at a density of 2 x 10 4 cells/cm 2 and cultured for 3 days (day 11). Here, another TGF-beta inhibitor, SB431542, was used as a negative control.
在第11天收集细胞，并计数细胞数量。使细胞悬浮于不含TGF-β抑制剂的培养基中，即用于增殖内皮细胞的培养基，并以2 x 104个细胞/cm2的密度接种至培养容器中。每1-3天更换培养基，并在第19天收集细胞，且计数细胞数量。同时，其一部分用于通过流式细胞术测量CD31 (一种内皮细胞标志物)阳性细胞比率。使剩余的细胞悬浮于用于增殖内皮细胞的培养基(不含TGF-β抑制剂)中，并然后将细胞以1 x 104个细胞/cm2的密度接种至培养容器中。每1-3天更换培养基，在第30天收集细胞，并计数细胞数量。同时，用流式细胞术测量CD31阳性细胞比率。Cells were collected on day 11 and the number of cells was counted. Cells were suspended in medium without TGF-beta inhibitor, ie, medium for proliferating endothelial cells, and seeded into culture vessels at a density of 2 x 104 cells/ cm2 . The medium was changed every 1-3 days and cells were harvested on day 19 and the number of cells was counted. Meanwhile, part of it was used to measure the CD31 (an endothelial cell marker) positive cell ratio by flow cytometry. The remaining cells were suspended in a medium for proliferating endothelial cells (without TGF-beta inhibitor), and the cells were then seeded into culture vessels at a density of 1 x 104 cells/ cm2 . The medium was changed every 1-3 days, the cells were harvested on day 30, and the number of cells was counted. Meanwhile, the ratio of CD31 positive cells was measured by flow cytometry.
当第8天的细胞数量定义为1时，第19天和第30天的细胞数量如表1所示。另外，第19天和第30天的CD31阳性细胞比率如表2所示。When the number of cells on day 8 was defined as 1, the number of cells on day 19 and 30 are shown in Table 1. In addition, the ratios of CD31-positive cells on the 19th day and the 30th day are shown in Table 2.
[表1][Table 1]
[表2][Table 2]
表明无论使用何种TGF-β抑制剂，第19天的CD31阳性率超过97%，使得通过本发明的方法获得了具有高纯度的血管内皮细胞。当使用SB431542时，第30天的细胞增殖率在10 μMSB431542中为0.9。另外，尽管第30天的细胞增殖率在20 μM SB431542中为5.1，但CD31阳性率为33.0%。另一方面，当RepSox用作TGF-β抑制剂时，第30天的细胞增殖率在含有3 μMRepSox的培养基中为2.5或更高和CD31阳性率为87%或更高。这些结果表明，与SB431542的那些相比较，RepSox在诱导血管内皮细胞的分化中可以高水平满足细胞增殖率和纯度两者，并且因此RepSox更有用。此处，由于在第11天在没有添加TGF-β抑制剂的培养基中未获得足够量的细胞，因此细胞未进行传代培养。It was shown that no matter what TGF-β inhibitor was used, the positive rate of CD31 on the 19th day was over 97%, so that the vascular endothelial cells with high purity were obtained by the method of the present invention. When SB431542 was used, the cell proliferation rate at day 30 was 0.9 in 10 μM SB431542. In addition, although the cell proliferation rate on day 30 was 5.1 in 20 μM SB431542, the CD31 positive rate was 33.0%. On the other hand, when RepSox was used as a TGF-β inhibitor, the cell proliferation rate on day 30 was 2.5 or higher and the CD31 positive rate was 87% or higher in the medium containing 3 μM RepSox. These results suggest that RepSox can satisfy both cell proliferation rate and purity at high levels in inducing differentiation of vascular endothelial cells compared to those of SB431542, and thus RepSox is more useful. Here, the cells were not subcultured since a sufficient amount of cells was not obtained on day 11 in the medium without the addition of a TGF-β inhibitor.
实施例2：血管内皮细胞的冷冻和解冻Example 2: Freezing and Thawing of Vascular Endothelial Cells
血管内皮细胞的诱导按照实施例1中所述的使用添加有20 μM RepSox的培养基的方法进行，并且从第18-22天收集细胞。洗涤收集的细胞，并悬浮于Stem Cell Banker (TAKARABIO INC.)中，使得细胞浓度为3 x 106/mL，并将1 mL的每种悬浮液分配于小瓶中。将小瓶置于冷冻处理的容器(BICELL, Nihon Freezer Co., Ltd.)中，并使细胞经受在保持于-80℃温度下的冷冻储存室中缓慢冷冻。之后，将每个小瓶转移至液氮中，并储存3天。Induction of vascular endothelial cells was performed as described in Example 1 using medium supplemented with 20 μM RepSox, and cells were harvested from days 18-22. The collected cells were washed and suspended in Stem Cell Banker (TAKARABIO INC.) so that the cell concentration was 3 x 106/mL, and 1 mL of each suspension was dispensed into vials. The vials were placed in freezer-treated containers (BICELL, Nihon Freezer Co., Ltd.) and cells were subjected to slow freezing in a freezer storage compartment maintained at a temperature of -80°C. After that, each vial was transferred to liquid nitrogen and stored for 3 days.
将冷冻的细胞用温热至37℃的水浴温热2分30秒以使细胞解冻。通过使用一部分细胞测量细胞成活力，并且结果是细胞成活力为87%。将全部量的剩余细胞加入预先分配有8 mL用于增殖内皮细胞的培养基的管中。之后，用1 mL用于增殖内皮细胞的培养基冲洗小瓶，并向管中加入冲洗过的培养基。将管以200 x g离心5分钟，并然后弃去上清液。将细胞悬浮于新鲜的用于培养内皮细胞的培养基中，并将细胞以2 x 104个细胞/cm2的密度接种至培养容器中。第二天更换培养基，并然后每1-3天更换培养基。Frozen cells were thawed by warming them in a water bath warmed to 37°C for 2 minutes 30 seconds. Cell viability was measured by using a fraction of cells, and as a result, cell viability was 87%. Add the entire amount of remaining cells to a tube pre-dispensed with 8 mL of medium for proliferating endothelial cells. Afterwards, rinse the vial with 1 mL of medium for proliferating endothelial cells and add the rinsed medium to the tube. The tubes were centrifuged at 200 xg for 5 minutes, and the supernatant was then discarded. The cells were suspended in fresh medium for culturing endothelial cells and seeded into culture vessels at a density of 2 x 10 4 cells/cm 2 . The medium was changed the next day and then every 1-3 days.
在解冻细胞之后第7天收集细胞，并计数细胞数量。同时，用流式细胞术测量CD31(一种内皮细胞标志物)阳性细胞比率。结果，发现细胞增殖率为1.5-4倍，和CD31阳性率为95%或更高(96.6-99%)。从结果可以看出，通过本发明的方法获得的内皮细胞通过冷冻和解冻具有非常小的损伤。Cells were harvested on day 7 after thawing and the number of cells was counted. At the same time, the CD31 (an endothelial cell marker) positive cell ratio was measured by flow cytometry. As a result, the cell proliferation rate was found to be 1.5-4 times, and the CD31 positive rate was 95% or higher (96.6-99%). As can be seen from the results, endothelial cells obtained by the method of the present invention have very little damage by freezing and thawing.
实施例3：CD31+/CD34+细胞的测量Example 3: Measurement of CD31+/CD34+ cells
按照培养基附带的说明书，在DEF-CS培养基中培养人iPS细胞(ChiPSC12品系)。Human iPS cells (ChiPSC12 strain) were cultured in DEF-CS medium according to the instructions accompanying the medium.
将TrypLE (商标) Select添加至已经传代培养的iPS细胞中，并将细胞在37℃下温育3-7分钟。通过吸液将分离的细胞收集为单细胞，并计数细胞数量。接下来，将细胞以6x 104个细胞/cm2的密度接种至涂有Matrigel (商标)的培养容器中，并在DEF-CS培养基中培养2-3天。之后，将培养基更换为添加有稀释至1:60的Matrigel的DEF-CS培养基，并将细胞再培养16-24小时，从而用Matrigel包被细胞的上层。TrypLE (trademark) Select was added to iPS cells that had been subcultured, and the cells were incubated at 37°C for 3-7 minutes. The detached cells were collected as single cells by pipetting and the number of cells was counted. Next, cells were seeded into Matrigel (trademark)-coated culture vessels at a density of 6×10 4 cells/cm 2 and cultured in DEF-CS medium for 2-3 days. After that, the medium was changed to DEF-CS medium supplemented with Matrigel diluted to 1:60, and the cells were cultured for an additional 16-24 hours to coat the upper layer of cells with Matrigel.
按照以下程序使获得的Matrigel包被的iPS细胞分化成中胚层细胞。首先，将Matrigel包被的iPS细胞的培养基更换为添加有100 ng/mL激活素A、2 mM GlutaMAX (商标)和B27补充剂(不含胰岛素)的RPMI 1640培养基(该时间点定义为开始诱导分化=第0天)，并将细胞培养24小时(第1天)。接下来，将培养基更换为添加有10 ng/mL hBMP4、10ng/mL hbFGF、2 mM GlutaMAX和B27补充剂的RPMI 1640培养基，并将细胞培养3天(第4天)。之后，将培养基更换为添加有100 ng/mL VEGF、2 mM GlutaMAX和B27补充剂的RPMI 1640培养基，并将细胞培养过夜(第5天)。此处，作为用光学显微镜观察所培养的细胞的结果，从第0天至第5天不能确认胚状体的形成。The obtained Matrigel-coated iPS cells were differentiated into mesoderm cells according to the following procedure. First, the medium of Matrigel-coated iPS cells was changed to RPMI 1640 medium supplemented with 100 ng/mL Activin A, 2 mM GlutaMAX (trademark), and B27 supplement (without insulin) (this time point was defined as Induction of differentiation started = day 0) and cells were cultured for 24 hours (day 1). Next, the medium was changed to RPMI 1640 medium supplemented with 10 ng/mL hBMP4, 10 ng/mL hbFGF, 2 mM GlutaMAX, and B27 supplement, and the cells were cultured for 3 days (day 4). After that, the medium was changed to RPMI 1640 medium supplemented with 100 ng/mL VEGF, 2 mM GlutaMAX and B27 supplement, and the cells were cultured overnight (day 5). Here, as a result of observing the cultured cells with an optical microscope, the formation of embryoid bodies could not be confirmed from the 0th day to the 5th day.
在第5天通过使用磁激活细胞分选术从培养的细胞中分离KDR阳性细胞，从而分离血管内皮祖细胞。为了分离KDR阳性细胞，将结合有PE荧光标记物的抗KDR抗体用作用于标记细胞的一抗，并用包埋抗PE抗体的磁珠收集用一抗标记的细胞。当用流式细胞术测量通过MACS分离的细胞中存在的KDR阳性细胞的比率时，KDR阳性比率为89-99%。将获得的细胞以2.7 x 104个细胞/cm2的密度接种至涂有Matrigel的培养容器中，并在添加有B27补充剂、50单位/mL青霉素、50 μg/mL链霉素、2 mM GlutaMAX、20 μM Y-27632和100 ng/mL VEGF的RPMI 1640培养基中培养16-24小时(第6天)。进一步地，将第6天的培养基更换为添加有B27补充剂、50单位/mL青霉素、50 μg/mL链霉素、2 mM GlutaMAX和100 ng/mL VEGF的RPMI1640培养基，并将细胞培养2天(第8天)。Vascular endothelial progenitor cells were isolated on day 5 by isolating KDR-positive cells from the cultured cells using magnetic activated cell sorting. To isolate KDR-positive cells, an anti-KDR antibody conjugated with a PE fluorescent marker was used as the primary antibody for labeling cells, and the cells labeled with the primary antibody were collected with anti-PE antibody-embedded magnetic beads. When the ratio of KDR-positive cells present in cells isolated by MACS was measured by flow cytometry, the KDR-positive ratio was 89-99%. The obtained cells were seeded at a density of 2.7 x 104 cells/ cm2 into Matrigel-coated culture vessels and were supplemented with B27 supplement, 50 units/mL penicillin, 50 μg/mL streptomycin, 2 mM GlutaMAX, 20 μM Y-27632 and 100 ng/mL VEGF in RPMI 1640 medium for 16-24 hours (day 6). Further, the medium on day 6 was changed to RPMI1640 medium supplemented with B27 supplement, 50 units/mL penicillin, 50 μg/mL streptomycin, 2 mM GlutaMAX, and 100 ng/mL VEGF, and the cells were cultured. 2 days (day 8).
在第8天收集细胞，并悬浮于添加有表3中列出的浓度的RepSox的用于增殖内皮细胞的培养基(Promocell)中。之后，将细胞以2.7 x 104个细胞/cm2的密度接种至培养容器中，并培养3天(第11天)。Cells were harvested on day 8 and suspended in Medium for Proliferating Endothelial Cells (Promocell) supplemented with RepSox at the concentrations listed in Table 3. After that, cells were seeded into culture vessels at a density of 2.7 x 10 4 cells/cm 2 and cultured for 3 days (day 11).
在第11天将培养基更换为不含TGF-β抑制剂的培养基，即用于增殖内皮细胞的培养基，并在第13天收集细胞，且计数细胞数量。此时，一部分细胞用于通过流式细胞术测量CD31阳性细胞和CD34阳性细胞。使剩余的细胞悬浮于用于增殖内皮细胞的培养基(不含RepSox)中，并且然后将细胞以2.7 x 104个细胞/cm2的密度接种至培养容器中。每1-3天更换培养基，并在第13天和第18天收集细胞，且计数细胞数量。同时，用流式细胞术测量CD31阳性细胞和CD34阳性细胞。On day 11, the medium was changed to medium without TGF-beta inhibitor, ie, medium for proliferating endothelial cells, and on day 13 cells were harvested and the number of cells was counted. At this time, a fraction of cells were used to measure CD31-positive cells and CD34-positive cells by flow cytometry. The remaining cells were suspended in medium for proliferating endothelial cells (without RepSox), and the cells were then seeded into culture vessels at a density of 2.7 x 10 4 cells/cm 2 . The medium was changed every 1-3 days, and cells were harvested on days 13 and 18, and the number of cells was counted. Meanwhile, CD31-positive cells and CD34-positive cells were measured by flow cytometry.
此处，CD31为内皮细胞的标志物，和CD34为造血干细胞或内皮祖细胞的标志物。另外，CD34也在幼年内皮细胞中表达。因此，本发明中的CD31阳性和CD34阳性细胞(下文中CD31+/CD34+)被认为是血管内皮细胞中尚未发展分化阶段的细胞，即幼年血管内皮细胞。第3天和第18天的CD31+/CD34+细胞比率如表3所示。Here, CD31 is a marker for endothelial cells, and CD34 is a marker for hematopoietic stem cells or endothelial progenitor cells. In addition, CD34 is also expressed in juvenile endothelial cells. Therefore, CD31-positive and CD34-positive cells (hereinafter CD31+/CD34+) in the present invention are considered to be cells that have not yet developed a differentiation stage among vascular endothelial cells, ie, juvenile vascular endothelial cells. The CD31+/CD34+ cell ratios on days 3 and 18 are shown in Table 3.
[表3][table 3]
无论RepSox的浓度如何，CD31+/CD34+细胞在第13天为95%或更大。另外，在第18天，随着RepSox的浓度变得更高，CD31+/CD34+细胞的比例变得更高。这些结果表明，在诱导血管内皮细胞的分化中，通过使用RepSox以高比例获得具有分化和成熟为各种内皮细胞的潜力的幼年内皮细胞。Regardless of the concentration of RepSox, CD31+/CD34+ cells were 95% or greater on day 13. In addition, on day 18, as the concentration of RepSox became higher, the ratio of CD31+/CD34+ cells became higher. These results suggest that in inducing the differentiation of vascular endothelial cells, juvenile endothelial cells with the potential to differentiate and mature into various endothelial cells are obtained at a high ratio by using RepSox.
工业应用性Industrial applicability
根据本发明，提供了高质量的内皮细胞。本发明的内皮细胞特别用于心肌层的产生。According to the present invention, high-quality endothelial cells are provided. The endothelial cells of the present invention are particularly useful for the generation of the myocardium.
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